Characterization of Almond Scion/Rootstock Communication in Cultivar and Rootstock Tissues through an RNA-Seq Approach

The rootstock genotype plays a crucial role in determining various aspects of scion development, including the scion three-dimensional structure, or tree architecture. Consequently, rootstock choice is a pivotal factor in the establishment of new almond (Prunus amygdalus (L.) Batsch, syn P. dulcis (Mill.)) intensive planting systems, demanding cultivars that can adapt to distinct requirements of vigor and shape. Nevertheless, considering the capacity of the rootstock genotype to influence scion development, it is likely that the scion genotype reciprocally affects rootstock performance. In the context of this study, we conducted a transcriptomic analysis of the scion/rootstock interaction in young almond trees, with a specific focus on elucidating the scion impact on the rootstock molecular response. Two commercial almond cultivars were grafted onto two hybrid rootstocks, thereby generating four distinct combinations. Through RNA-Seq analysis, we discerned that indeed, the scion genotype exerts an influence on the rootstock expression profile. This influence manifests through the modulation of genes associated with hormonal regulation, cell division, root development, and light signaling. This intricate interplay between scion and rootstock communication plays a pivotal role in the development of both scion and rootstock, underscoring the critical importance of a correct choice when establishing new almond orchards.


Introduction
In modern orchards, rootstocks serve a dual purpose of selecting specific root system traits and conferring desirable agronomic traits to trees and fruits [1,2].These effects on scion development have been extensively documented across various tree species, ranging from tree vigor to yield or fruit quality [1,[3][4][5][6].Vigor and architecture in particular are important for high-density planting systems.Recently, molecular approaches have been conducted in woody plant species to elucidate the underlying mechanisms at the molecular level [7,8].However, the analysis of the scion effect on the rootstock has been limited to the graft formation, examining the processes occurring during the vascular union, which results in vascular regeneration and the establishment of the graft junction [9,10].However, little is known about how the scion can influence the phenotypic traits exhibited by the rootstock, including nutrient assimilation, pathogen resistance, and root development [11].These traits may be influenced in a variable manner depending on the specific scion cultivar that is grafted onto the rootstock.
Rootstock development is regulated by various phytohormones, participating in the regulation of cell elongation, cell division, and cell differentiation [12,13].Like the aerial part of the plant, auxin plays a vital role in governing various processes in roots, including root patterning, cell division, and cell elongation [14][15][16].Strigolactones (SLs) act in conjunction with auxin, exerting control over lateral root formation and root-hair elongation, while mediating root responses to environmental changes [17][18][19].Cytokinins (CKs) promote cell differentiation and cell division in different root tissues and counteract auxin by inhibiting lateral root formation [20][21][22][23].Gibberellic acid (GA) contributes to the maintenance of root cell proliferation and cell elongation in the meristem, while suppressing lateral root formation [24][25][26].Brassinosteroids (BRs) play a crucial role in regulating root meristem activity and also participate in the regulation of lateral root initiation and root cell elongation [27,28].Ethylene (ET) modulates meristem maintenance, promoting cell division and acting in opposition to auxin in lateral root formation [29,30].
Light signaling exerts significant control over plant development through diverse mechanisms.In plants, the circadian clock regulates various developmental processes in response to fluctuations in exposure to light, including seed germination, hypocotyl elongation, root growth, and flowering [31,32].The regulation of the circadian clock is also intertwined with carbohydrate metabolism and nutrient assimilation [33].Furthermore, the shade avoidance response plays a role in regulating plant growth, which depends on the ratio of red light to far-red light (R:FR) detected by phytochrome photoreceptors phyA and phyB.Alterations in this ratio elicit changes in the auxin flux, thereby influencing the direction and activity of plant growth [34][35][36][37][38].
In this study, we carried out a transcriptomic analysis of the responses associated with both the rootstock influence on the scion and the scion influence on the rootstock.To investigate these interactions, we grafted two almond commercial cultivars characterized by contrasting architecture and vigor traits onto two almond × peach (Prunus amygdalus (L.) Batsch, syn P. dulcis (Mill.).× P. persica (L.) Batsch).This resulted in a total of four combinations.Our goal was to elucidate the biological processes and molecular responses that were specifically affected in both the upper and lower regions of the graft site.

'Isabelona' and 'Lauranne' Vigor Was Influenced by the Rootstock
Data on tree architecture were obtained for the four graft combinations, 'Isabelona'/Garnem ® , 'Isabelona'/'GN-8', 'Lauranne'/Garnem ® , and 'Lauranne'/'GN-8' (Figure 1).As the trees were still in their early stages and had not yet developed branches, measurements were restricted to trunk length (Length) and the diameters of both the scion (d_Scion) and the rootstock (d_Rootstock).Due to the inherent challenges associated with accurately measuring root architecture, no data were collected on the root architecture.
Combinations involving 'Lauranne' as the scion displayed higher Length values (Table 1, Figure 1).Regarding the rootstocks, the use of the vigor-conferring rootstock Garnem ® resulted in higher length values for both cultivars, presenting higher values than when grafted onto the dwarfing rootstock 'GN-8' (Table 1, Figure 1).Values within columns for each scion/rootstock combination followed by the same letter were not significantly different (p > 0.05).

Rootstock Only Influenced Gene Expression in Combinations with 'Isabelona
A transcriptomic analysis from cultivar samples was carried out to v their gene expression was affected by the rootstock genotype during the development (Table S1).A Principal Component Analysis (PCA) was co gene expression data from the four combinations, with each gene serving The first component (PC1; 83.5%) accounted for most of the variability ( samples were primarily arranged by their respective cultivars, while the r Trunk diameter (d_Scion), often represented as Trunk Cross Sectional Area (TCSA), is commonly used as a measure of tree vigor.Similar to the length values, 'Lauranne' exhibited higher d_Scion values than 'Isabelona'.Additionally, when grafted onto Garnem ® , both cultivars displayed higher d_Scion values compared to grafting onto 'GN-8' (Table 1).However, no significant difference was observed in the diameters of the rootstocks (d_Rootstock), although the mean values were slightly lower when 'Isabelona' was used as the scion (Table 1).

Rootstock Only Influenced Gene Expression in Combinations with 'Isabelona'
A transcriptomic analysis from cultivar samples was carried out to verify whether their gene expression was affected by the rootstock genotype during the early stages of development (Table S1).A Principal Component Analysis (PCA) was conducted using gene expression data from the four combinations, with each gene serving as a variable.The first component (PC1; 83.5%) accounted for most of the variability (Figure 2).The samples were primarily arranged by their respective cultivars, while the rootstock genotype had a minimal impact on their distribution.Notably, no DEGs were detected for the 'Lauranne' combinations.In contrast, 221 DEGs were identified in the combinations involving 'Isabelona' as the scion (Table S1).
Combinations of 'Isabelona' grafted onto the vigor-conferring rootstock Garnem ® resulted in notable alterations in gene expression levels associated with auxin regulation, predominantly through repressive mechanisms.Furthermore, distinct expression patterns were observed for genes involved in stress hormone signaling, specifically ABA and ET pathways.Moreover, DEGs related to plant development, cell wall reorganization, and the regulation of growth-related hormones GA and BRs were identified (Table 2).Combinations of 'Isabelona' grafted onto the vigor-conferring rootstock Garnem ® resulted in notable alterations in gene expression levels associated with auxin regulation, predominantly through repressive mechanisms.Furthermore, distinct expression patterns were observed for genes involved in stress hormone signaling, specifically ABA and ET pathways.Moreover, DEGs related to plant development, cell wall reorganization, and the regulation of growth-related hormones GA and BRs were identified (Table 2).The cultivar effect of commercial almond cultivars 'Lauranne' and 'Isabelona' on the rootstock transcriptome was analyzed in a vigorous rootstock like Garnem ® , and a dwarfing rootstock such as 'GN-8'.We carried out a PCA using the expression of each gene as variables for the four different scion/rootstock combinations.The first two components explained 51.5% of the variability, while none of the other variables explained more than 14%.PC1 and PC2 explained 28.8% and 22.7% of the variability, respectively.In the PCA, there was a clear separation between the four different combinations (Figure 3A).Combinations with Garnem ® as rootstock are in the lower-left corner while combinations with 'GN-8' are in the upper-right corner.Therefore, there is a clear effect of the rootstock, and it can be observed in the gene expression, with individuals clearly segregating depending on which scion, 'Lauranne' or 'Isabelona', is grafted onto them (Figure 3A).A total of 36 genes were only differentially expressed in combinations with 'GN-8' as rootstock, while 342 DEGs were identified in combinations with Garnem ® .Both rootstocks shared 28 DEGs (Figure 3B).Therefore, while the number of DEGs was considerably more elevated when comparing combinations that had been grafted onto the vigor-conferring rootstock Garnem ® , than those grafted onto the dwarfing rootstock 'GN-8', around half of DEGs found in the later were shared between both rootstocks.

Garnem ® Transcriptome Is More Affected by Cultivar Effect Than the 'GN-8' Transcriptome
The rootstock exhibited varying degrees of cultivar influence.In the case of 'GN-8', a limited number of DEGs were observed, indicating a relatively minor impact on gene expression.Conversely, the rootstock Garnem ® displayed a higher degree of differential expression, suggesting a more pronounced influence on the transcriptome (Figure 3B).Among the limited number of genes that showed differential expression in 'GN-8', several have homologues known to participate in plant development, such as CLV1 (Prudul26A015064), FIP37 (Prudul26A020498), EF1A (Prudul26A027600), and VIM1 (Prudul26A008660).Furthermore, specific to root development, homologues of genes such as IAMT1 (Prudul26A029624) and DRMH3 (Prudul26A007496) were found to be less expressed in combinations where 'Lauranne' served as the cultivar (Table 3).Moreover, genes involved in hormonal response or transport, including homologues of LAX3 (Prudul26A031524) and RD22 (Prudul26A001972), also exhibited lower expression levels when 'Lauranne' was grafted onto the 'GN-8'.

Garnem ® Transcriptome Is More Affected by Cultivar Effect than the 'GN-8' Transcriptome
The rootstock exhibited varying degrees of cultivar influence.In the case of 'GN-8', a limited number of DEGs were observed, indicating a relatively minor impact on gene expression.Conversely, the rootstock Garnem ® displayed a higher degree of differential expression, suggesting a more pronounced influence on the transcriptome (Figure 3B).Among the limited number of genes that showed differential expression in 'GN-8', several have homologues known to participate in plant development, such as CLV1 (Prudul26A015064), FIP37 (Prudul26A020498), EF1A (Prudul26A027600), and VIM1 (Prudul26A008660).Furthermore, specific to root development, homologues of genes such as IAMT1 (Prudul26A029624) and DRMH3 (Prudul26A007496) were found to be less expressed in combinations where 'Lauranne' served as the cultivar (Table 3).Moreover, The impact of the cultivar on the transcriptome of the Garnem ® rootstock was found to be more pronounced, influencing multiple pathways associated with plant development.Several homologues of genes involved in auxin response, such as IAA4 (Prudul26A024452) and SAUR50 (Prudul26A025556), as well as auxin transport, like LAX3 (Prudul26A031524), exhibited differential expression, with lower expression levels observed when 'Lauranne' was used as the scion (Table 4).Furthermore, genes involved in other hormonal regulatory pathways, including ABA, BR, CK, and ET response, as well as GA biosynthesis, were also identified as differentially expressed (Table 4).Specifically, homologues of genes related to ET response, such as EGY3 (Prudul26A021691) and ERF24 (Prudul26A019984), displayed increased expression in combinations where 'Lauranne' served as the scion.Con-versely, homologues of BR response genes, including TINY2 (Prudul26A010146) and BAS1 (Prudul26A030744), exhibited lower expression levels in combinations with 'Lauranne' as the scion.The cultivar genotype exerted a significant influence on growth-related processes, impacting cell division, cell wall reorganization, and organ development (Table 4).Homologues of genes involved in the cell division complex, such as MCM genes (Prudul26A029769, Prudul26A013222) and RCC1 (Prudul26A003343, Prudul26A018495), displayed overexpression when 'Lauranne' was the cultivar.Similarly, homologues of genes with crucial roles in plant development, such as GI (Prudul26A016707) and PAT1 (Prudul26A024652), were also found to be overexpressed when 'Lauranne' acted as the scion.Conversely, multiple other homologues of genes involved in various functions related to plant development, including MAX1 (Prudul26A022418) and FIP37 (Prudul26A020498), exhibited lower expression levels in combinations with 'Lauranne' as the cultivar.Notably, root development was also affected, with genes such as AGL79 (Prudul26A020939) and DRMH3 (Prudul26A007496) presenting reduced expression in combinations with 'Lauranne'.

Discussion
Prior research has established that the rootstock genotype has the capacity to induce phenotypic alterations in the tree architecture of almond cultivars [39,40].Nevertheless, the precise mechanisms underlying this intricate interplay between the scion and rootstock at the molecular level have yet to be comprehensively elucidated.In this study, we analyzed how two hybrid rootstocks ('GN-8' and Garnem ® ) modulate the transcriptome of two distinct almond commercial cultivars ('Isabelona' and 'Lauranne').Additionally, we explored how these cultivars might reciprocally influence the transcriptome of the respective rootstocks.
'Isabelona' has previously been described to exhibit reduced vigor and strong apical dominance, resulting in a phenotype characterized by limited branching and elongated trunks.Conversely, 'Lauranne' has been reported to display high vigor and weak apical dominance, leading to extensive branching and shorter trunks [39,40].In the current study, the trees are in their initial year of growth, and they have not yet developed branches that compete with the main axis growth.Consequently, the increased vigor observed in the 'Lauranne' cultivar may contribute to the higher length values reported (Table 1).Ultimately, the observed differences in phenotype appear to predominantly hinge on the vigor displayed by each graft combination.However, it is plausible that the biological processes governing the development of specific tree architectures for each combination have already been established, despite not yet being visually discernible in these one-yearold plants.
The rootstock genotype had a restricted impact on the transcriptome of the studied cultivars (Figure 2; Table S2).Whereas a limited number of DEG was discerned in combinations with 'Isabelona' as the cultivar, no DEGs were identified when 'Lauranne' was the scion.This observation reaffirms the limited malleability of these specific cultivars by the rootstock genotype, corroborating earlier findings [39].This characteristic underscores the potential utility of these cultivars as an ideal platform for exploring whether the cultivar genotype can exert a modulatory effect on the expression profile of the rootstock.
In terms of the rootstock's influence on 'Isabelona', various genes related to plant development displayed differential expression patterns (Table 2).Combinations in which Garnem ® served as the rootstock exhibited an upregulation of genes associated with auxin regulation, likely in a repressive manner [41][42][43][44], indicating a potential reduction in apical dominance [45,46].Auxin-related genes such as Aux/IAA can be found in phloem and might actually be synthetized in the rootstock: Lower expression of IAA4 in persimmon induced vigor [47], which is consistent with our observation.As it appears downregulated in both the rootstock and the scion when Laurane is grafted with Garnem ® this RNA might be mobile.Additionally, DEGs involved in processes linked to active growth, such as cell proliferation and cell expansion [48][49][50], as well as genes promoting nitrogen assimilation [51][52][53], were upregulated in 'Isabelona'/Garnem ® combinations.This upregulation likely contributed to the enhanced vigor observed in scions grafted onto Garnem ® (Figure 1; Table 1).Furthermore, genes that positively regulate stress responses and growth inhibition were upregulated when 'GN-8' was used as the rootstock [54][55][56], which may be linked to the limited cultivar growth caused by this dwarfing rootstock.
Given that distinct rootstocks have the capacity to influence almond scion architecture, potentially altering branch number and main axis growth, it is reasonable to consider that the grafted scion, in turn, exerts an influence on the rootstocks, triggering diverse mechanisms that could impact rootstock properties.This reciprocal effect has previously been documented in other plant species with respect to various traits, including the regulation of rootstock responses to low phosphorus (Pi) conditions and phloem sap metabolites [57,58].In the present study, it was observed that the cultivar genotype did indeed influence the rootstock transcriptome (Figure 3).However, the extent of this influence was notably contingent on the specific rootstock employed.Although our samples were collected from the rootstock trunk, it is conceivable that the alterations in the transcriptome dynamics observed there may have broader ramifications for the entire root system.
The rootstock 'GN-8' exhibited a significantly lower number of DEGs compared to Garnem ® (Figure 3B), although approximately half of these DEGs were shared between the two rootstocks.Consequently, it appears that the cultivar effect on both rootstocks follows a similar direction, with the key distinguishing factor being the magnitude of this effect.The diminished intensity of the influence on the 'GN-8' transcriptome could potentially be attributed to the dwarfing characteristics associated with this particular rootstock.Such dwarfing rootstocks are often characterized by less active metabolic processes, which could render them less susceptible to modulation by the scion's influence.This may elucidate the observed reduction in the extent of transcriptomic alteration in 'GN-8' as compared to Garnem ® .

Plant Material and Growth Conditions
For the experimental setup, two almond commercial cultivars, 'Isabelona' and 'Lauranne' were grafted onto two distinct hybrid rootstocks known as Garnem ® (a commercially available rootstock) and 'GN-8' (a newly selected accession), obtaining four different combinations.Both rootstocks are almond × peach (P.amygdalus (L.) Batsch, syn P. dulcis (Mill.).× P. persica (L.) Batsch) hybrid rootstocks.The selection of 'Isabelona' and 'Lauranne' cultivars was based on their reduced influence exerted by the rootstock on their apical dominance and branch formation phenotypes [39].Grafted plants were supplied by the Agromillora Iberia S.L. (Barcelona, Spain) nursery in 2020.Subsequently, the plants were temporarily housed in a nursery before sample collection, which took place at the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA).Standard nursery practices were maintained during the experimental period.

Phenotypic Data Collection in Nursery
Phenotypic data were collected for ten replicates of each of the four combinations, prior to sample collection.Three vigor-related parameters were measured: scion axis length (Length), scion trunk diameter (d_Scion), and rootstock trunk diameter (d_Rootstock).The length measurement was determined from the graft union.To quantify d_Scion and d_Rootstock, a caliper was employed to measure 20 mm above and 20 mm below the graft union, respectively.

RNA-Seq Analysis
During the summer of 2020, samples were collected from nine (three per sample) different individuals per combination, encompassing a region 50 mm below and above the graft union.RNA extraction was performed on these samples utilizing the CTAB method, as previously described, with modifications [88][89][90].Stranded mRNA-Seq analysis was conducted at the Centro Nacional de Análisis Genómico (CNAG-CRG) in Barcelona, Spain.Sequencing was performed using the Illumina NovaSeq 6000 System, generating > 30 M PE reads per sample and a read length of 2 × 50 bp.The resulting FASTQ files were processed with FASTQ Groomer (Galaxy Version 1.1.1)[91].Adapter sequences were subsequently removed from the reads using Trimmomatic (Galaxy Version 0.38.0)[91,92].Alignment of the RNA-Seq data was performed using HISAT2 (Galaxy Version 2.2.1), employing a maximum intron length of 20,000 bp [93] and aligning to the P. dulcis 'Texas' Genome v2.0 [94].Duplicated molecules were identified using the MarkDuplicates tool (Galaxy Version 2.18.2.2), and mate-pair information was confirmed using FixMateInformation (Galaxy Version 2.18.2.1) Picard tools (http://broadinstitute.github.io/picard,accessed on 30 November 2022).Gene expression was quantified using featureCounts (Galaxy Version 2.0.1+galaxy2)[95] using the P. dulcis 'Texas' Genome v2.0 gene annotation, which comprises 27,044 genes (https://www.rosaceae.org/analysis/295,accessed on 30 November 2022).Differential analysis of the count data was conducted using edgeR (Galaxy Version 3.36.0)with default settings [96], considering genes with a corrected p-value below 0.05 and a log2 fold change (log2FC) greater than 0.75 or less than −0.75 as differentially expressed.All procedures were performed using the Galaxy platform.Recent studies have demonstrated the robustness of RNA-Seq methods, with validation through qPCR confirming results for most transcripts.Discrepancies between the two techniques were observed in less than 2% of cases, primarily involving short and low-expressed genes [97,98].As this data relates to Fragments Per Kilobase of transcript per Million (FPKM), differentially expressed genes were filtered to ensure they belonged to the top 98% of transcripts with the highest FPKM values.

Statistical Analysis
All statistical analyses were carried out in the R platform (https://cran.r-project.org/, accessed on 30 November 2022).Significant differences in phenotypic data were evaluated using an ANOVA test.These were assessed with a Tukey's test (p < 0.05) using the agricolae R package (https://CRAN.R-project.org/package=agricolae,accessed on 30 November 2022).Principal component analysis (PCA) was conducted using R stats package with default parameters on the gene expression values for the all the genes in the four combinations.

Conclusions
The interaction between scion and rootstock in almond trees is bidirectional, exerting influence on the development of both the scion and the rootstock.In this study, we have identified several biological processes that undergo differential modulation depending on the rootstock genotype, impacting various aspects of cultivar growth and development.Additionally, this influence appears to produce a feedback mechanism within the rootstock developmental processes.Specifically, our findings demonstrate that cultivars displaying heightened vigor, exemplified by 'Lauranne', have a positive influence on root development.This positive impact enhances the radicular system's capacity to efficiently absorb nutrients from the soil.Consequently, this nurtures scion growth, ultimately resulting in the robust and vigorous phenotype exhibited by 'Lauranne' when compared to 'Isabelona'.Hence, the selection of an appropriate scion/rootstock combination is a pivotal determinant for the success of almond orchards.In intensive cultivation systems, the rootstock impact on tree vigor depends not only on its genotype but also on the complementary interactions with the scion.It is in this synergy that root development is optimized, consequently influencing overall tree growth and productivity.

Data Availability Statement:
The datasets generated and/or analyzed during the current study are available in the European Nucleotide Archive (https://www.ebi.ac.uk/ena/browser/home, accessed on 30 July 2021) and are accessible through the accession number PRJEB50411.

Table 1 .
Analysis of architectural traits related to vigor in one-year-old scion/rootstock combinations.

Table 1 .
Analysis of architectural traits related to vigor in one-year-old scion/roo tions.Cultivar Rootstock Length (mm) d_Scion (mm) d_Roo